Gelatin is the denatured derivative of collagen, a protein containing 18 kinds of amino acids necessary for human body. Natural gelatin is usually prepared by acidic or alkalic hydrolysis of collagen in connective tissues e.g. animal skin, bone, sinew and ligament etc., separation and then extraction. Usually it can be divided A and B two types: the one prepared through acidic hydrolysis, with less side chain carboxylic acid residual group and higher isoelectric point, is called as A type; the one prepared through alkali hydrolysis is called as B type, with most glutamine and asparagine residue in side chains being transformed into glutamic acid and aspartic acid, therefore the side chains have more carboxylic acid residual group and it has lower isoelectric point. Natural gelatin, whether prepared by acidic method or alkalic method, is usually a complicated mixture, with many polypeptide of different molecular weight. Its property differs to some extent according to different batches. Gelatin can also be prepared through genetic recombination engineering, and the gelatin by this method has precise molecular weight, isoelectric point, and molecular structure which can be designed according to specific application (Olsen et al, Advanced Drug Delivery Reviews, 55, 1547, 2003).
Gelatin has many excellent properties such as biocompatibility, biodegradation and low immunogenicity etc. Therefore, it has wide usages in bio-medicine fields such as gelatin hemostasis sponge, gelatin capsule for drug, gelatin sponge wound dressing and new drug release formulation and tissue regeneration matrix etc. However, gelatin can dissolve into water at body temperature; therefore for the most applications of gelatin in bio-medicine, physical crosslinking and chemical crosslinking of gelatin to improve its thermal and mechanical stability is necessary. Dehydrogenation heat treatment and UV radiation are common methods for physical crosslinking, but their crosslinking efficacy is low and also uncontrollable. Chemical crosslinking has relatively higher efficacy. The common chemical crosslinkers include formaldehyde, glutaric dialdehyde, polyfunctional group epoxy crosslinker, polyfunctional group isocyanate crosslinker, acid azid diazoimido compounds and carbodiimide etc. (Kuijpers et al, Journal of Biomaterials Science: Polymer Edition, 11, 225, 2000). However, these chemical crosslinkers usually have severe toxic side effects, and even trace residue of chemical crosslinker and crosslinking functional groups may lead to severe inflammation. Therefore, the application of gelatin in bio-medicine field is seriously restricted by the current available chemical crosslinking method.
Chemical modification of gelatin is one important way to improve the application of gelatin in bio-medicine field, which can not only offer gelatin some important physical and chemical properties, but also reduce or not use the chemical crosslinker with toxic side effect in making crosslinked gelatin materials. For example, Ma et al disclosed a hydrophobically poly-lactic acid modified gelatin derivative with amphipathic properties in Journal of Biomaterials Science: Polymer Edition, 13, 67, 2002; Van Den Bulcke et al disclosed methylacrylamide modified gelatin derivative in Biomacromolecules, 1, 31, 2000, and this derivative can be used to prepare crosslinked gelatin material by free radical polymerization through relatively mild light-induction; Morikawa and Matsuda et al disclosed a N-isopropylacrylamide grafted gelatin derivative with temperature-responsive gelation property in Journal of Biomaterials Science: Polymer Edition, 13, 167, 2002; the thiolated gelatin derivative disclosed by Shu et al in Biomaterials, 24, 3825, 2003, can be used to prepare in situ crosslinked material through mild disulfide bond or nucleophilic addition method. So far, these methods still have many limits in preparing crosslinked gelatin material. Herein one important reason is the number of main functional groups of gelatin (amino group or carboxyl in side chain) available for chemical modification and crosslinking is considerably limited. For example, for type B gelatin, there is 28 side chain amino group and 118 side chain carboxyl per 1000 amino acid residual groups. There are similar side chain amino group content in terms of type A gelatin and type B gelatin, however, the content of side chain carboxyl in type A gelatin (54/1000 amino acid residues) which is far less than that in type B gelatin. Therefore, it is necessary to further develop novel method of chemical modification and crosslinking to further expand many kinds of application potential of gelatin in biomedicine field.
Invention Contents:
One of technical problems to be resolved in this invention is to provide a kind of novel multiple modified derivatives of gelatin.
The other technical problem to be resolved in this invention is to provide a kind of novel crosslinked material made of multiple modified derivatives of gelatin.
In this invention gelatin is taken as raw material, and the novel multiple modified derivatives of gelatin is prepared by conducting hydrophobic modification on the side chain amino group of gelatin through amide bond, carboxylation on side chain amino group through the amide bond and then thiolation of carboxyl. Compared with the single modified derivatives of gelatin, the multiple modified derivatives of gelatin of this invention have many excellent properties such as adjustable side chain molecular structure and chemical properties etc., and may have many important applications in bio-medicine field.
The multiple modified derivatives of gelatin of this invention has undermentioned general formula structure (I) and at least one of the general formula structure (II), (III), and (IV) at the same time.

Wherein G is a gelatin residue, including type A gelatin residue, type B gelatin residue, or a gelatin obtained from genetic recombination. R1 is an alkylidene or a connection group with an amide bond. R2 is an alkyl or an aryl. R3 is alkylidene or a substituted alkylidene. R4 is carboxyl or carboxyl salt.
The above mentioned connection group containing one amide bond is
Wherein R′ and R″ are alkylidene group, substituted alkylidene, aryl or polyether group.
The above mentioned alkylidene group is —(CH2)n— (n is an integer from 1 to 15). Preferably n is an integer from 1 to 8
The above mentioned substituted alkylidene is an alkylidene group that one of its hydrogen atoms at least is substituted by alkyl, hydroxyl, amino, alkoxy, phenyl, ester group etc.
The above mentioned aryl is aromatic phenyl or naphthyl etc., and preferably phenyl.
The above mentioned polyether group is —[(CHR)nO]m, wherein R is an alkyl, n is an integer from 1 to 10, and m is an integer from 1 to 500. Preferably R is hydrogen atom, and n equal to 2, 3 and 4, respectively.
The above mentioned alkyl is a linear chain alkyl or a fork chain alkyl with 1˜15 carbon atoms e.g. methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, sec-butyl, amyl, neoamyl, hexyl, heptyl, octyl etc., and preferably linear chain or fork chain alkyl containing 1˜10 carbon atoms, especially preferably methyl, ethyl, propyl, butyl, amyl, hexyl, heptyl and octyl.
The above mentioned alkoxyl is linear chain or fork chain alkoxyl containing 1˜6 carbon atoms e.g. methoxyl, ethoxyl, propoxy, isopropoxy, butoxy, isobutoxy, tert-butoxy, sec-butoxy, pentyloxy, neo-pentyloxy and hexyloxy etc, and preferably linear chain or fork chain alkoxyl containing 1˜4 carbon atoms, especially preferably methoxyl and ethoxyl.
The above mentioned ester group is —C(O)OR, wherein R is low alkyl group above mentioned, and preferably carbomethoxy, carbethoxy, propyl ester group and butyl ester group.
The above mentioned carboxyl is —COOH. Carboxyl salt is the group resulting from the above mentioned carboxyl neutralized by alkali i.e. —COO31A+, wherein A+contains sodium, potassium ion, lithium ion and ammonium ion etc., and preferably carboxyl, carboxyl sodium salt or carboxyl potassium salt.
The multiple modified derivatives of gelatin of this invention has following several kinds of representative chemical structural general formulas:

Wherein, the definition of G, R1, R2, R3 and R4 are the same as before.
The preferable chemical structures of R1, R2, R3 and R4 in the multiple modified derivatives of gelatin of this invention are as follows:

The multiple modified derivatives of gelatin in the above mentioned general formulas from (V) to (XI) all contain at least one thiol. General formulas (V), (VI), and (VII) are the chemical structure formula of double modified derivatives of gelatin of this invention. General formulas (VIII), (IX) and (X) are the chemical structure formula of triple modified derivatives of gelatin of this invention. General formula (XI) is the chemical structure formula of quadruple modified derivatives of gelatin of this invention.
The chemical modification of this invention on the multiple modified derivatives of gelatin contains the following four modes: (A) hydrophobic modification on the side chain amino group of gelatin through amide bond; (B) carboxylation on the side chain amino group of gelatin through amide bond; (C) thiolation on the side chain carboxyl of gelatin; (D) thiolation after carboxylation on the amino group of gelatin through amide bond.
For chemical modification mode (A), the commonly used method for hydrophobic modification on the side chain amino group of gelatin through amide bond is as follows:

Wherein, the definition of G and R2 are the same as previously. The detailed commonly used preparation process is to dissolve gelatin in warm water to make aqueous solution (usually at 30° C.), then adjust the pH value of solution to alkalescence (usually 8˜10). After that, add in anhydride, stir to react for a certain time, and at the same time add in appropriate amount of aqueous alkali solution (e.g. sodium hydroxide) to maintain the reaction solution as alkalescence. Finally, dialyze and purify the reaction solution, freeze drying to get the product. The adopted anhydride contains acetic anhydride, propionic anhydride, butyric anhydride, valeric anhydride, caproic anhydride, heptanoic anhydride and octanoic anhydride etc.
For chemical modification mode (B), the commonly used method for carboxylation on the side chain of gelatin through amide bond is as follows:

The detailed commonly used preparation process is to dissolve gelatin in warm water to make aqueous solution (usually at 30° C.), then adjust the pH value of solution to alkalescence (usually 8˜10). After that, add in diacid anhydride, stir to react for a certain time, and at the same time add in appropriate amount of aqueous alkali solution (e.g. sodium hydroxide) to maintain the reaction solution as alkalescence. Finally, dialyze and purify the reaction solution, freeze drying to get the product. The adopted diacid anhydride contains butanedioic anhydride, glutaric anhydride, hexanedioic anhydride, maleic anhydride, heptanedioic anhydride, and octandioic anhydride etc.
For chemical modification mode (C) and (D), the chemical method of hydrazide/carbodiimide coupling (Shu et al, Biomacromolecules, 3, 1304, 2002) is adopted to conduct thiolation on the side chain carboxyl of gelatin (includes the introduced carboxyl from carboxylation on the side chain amino group of gelatin through amide bond). Its fundamental principle is that, the side chain carboxyl of gelatin (or the introduced carboxyl from carboxylation on the side chain amino group of gelatin through amide bond) produces reactive intermediate under the activation of carbodiimide, then the hydrazide amino of dithio-dihydrazide react with the reactive intermediate by nucleophilic attack to produce affixture, finally the disulfide bond of the affixture is reduced into free thiol, and then the product is collected after purification. Wherein the commonly used method is as follows:

Wherein, the definitions of G and R1 are the same as previously. The detailed commonly used preparation process is to dissolve gelatin or the carboxylation modified derivatives of gelatin into warm water to make aqueous solution (usually at 30° C.), then adjust the pH value of solution to subacidity (usually 4.75). After that, add into a certain dithio-dihydrazide, stir and dissolve it, then add a certain amount of 1-ethyl-3-(3-dimethylamino propyl)carbodiimide hydrochloride, and at the same time add into appropriate amount of acid solution continuously (e.g. hydrochloric acid) to maintain the pH value of reaction solution at 4.75; Finally under the condition of alkalescence (usually the pH value is 8˜10), add into reducer such as hydroxy thiol, dithiothreitol or sodium borohydride etc. to reduce the disulfide bond into free thiol, the impurities are removed by dialysis and purification under acid condition, and freeze drying to get the product.
With regard to chemical modification way (C) and (D), for the thiolation on the side chain carboxyl of gelatin (or the introduced carboxyl from carboxylation on the side chain amino group of gelatin through amide bond), the commonly adopted representative dithio-dihydrazides include dithiodipropionic dihydrazide (DTPDH) and dithiodibutanoic dihydrazide (DTBDH) published in Biomacromolecules, 3, 1304, 2002 by Shu et al., and the new dihydrazide compounds published in invention patent application by us (Chinese patent application number: 200610118715.2; invention name: dihydrazide compounds and preparation method and usage thereof), including dithiodipropionic diacyl glycine dihydrazide (abbr. DGDTPDH), dithiodipropionic diacyl alanine dihydrazide (abbr. DADTPDH), dithiodipropionic diacyl(hydroxyl)aminoacetic dihydrazide (abbr. DHADTPDH), dithiodipropionic diacyl aminopropionic dihydrazide (abbr. DPDTPDH), dithiodipropionic diacyl aminobutyric dihydrazide (abbr. DBDTPDH), di-dipropionic diacyl cystamino dihydrazide (abbr. DPCDH), disuccinic diacyl cystamino dihydrazide (abbr. DSCDH), di(methyl)-succinic diacyl cystamino dihydrazide (abbr. DMPCDH), diglutaric diacyl cystamino dihydrazide (abbr. DGCDH), and diadipic diacyl cystamino dihydrazide (abbr. DACDH) etc. The chemical structural formula of these representative dithio-dihydrazides are as follows:

The preparation of multiple modified derivatives of gelatin represented by above mentioned general formula (V) includes two processes: chemical modification mode (A) i.e. hydrophobic modification on the side chain amino groups of gelatin through amide bond, and chemical modification mode (C) i.e. thiolation on the side chain carboxyl of gelatin. The commonly adopted preparation method is divided into two steps: (1) to dissolve gelatin in warm water to make aqueous solution (usually at 30° C.), then adjust the pH value of solution to alkalescence (usually 8˜10). After that, add in anhydride, stir to react for a certain time, and at the same time add in appropriate amount of aqueous alkali (e.g. sodium hydroxide) to maintain the reaction solution as alkalescence. Finally, dialyze and purify the reaction solution, freeze drying to get the intermediate; (2) to dissolve the intermediate in warm water to make aqueous solution or to use the above unpurified intermediate solution directly (usually at 30° C.), then adjust the pH value of solution to subacidity (usually 4.75). After that, add in a certain amount of dithio-dihydrazide, stir and dissolve it, and then add a certain amount of 1-ethyl-3-(3-dimethylamino propyl) carbodiimide hydrochloride, and at the same time add into appropriate amount of acid solution continuously (e.g. hydrochloric acid) to maintain the pH value of reaction solution at 4.75; Finally under the condition of alkalescence (usually the pH value is 8˜10), add in reducing agents such as hydroxyl thiol, dithiothreitol or sodium borohydride etc. to reduce the disulfide bond into free thiol, the impurities are purified by dialysis under acid condition. Freeze drying to get the multiple modified derivatives of gelatin represented by general formula (V) of this invention. The adopted acid anhydride include acetic anhydride, propionic anhydride, butyric anhydride, pentanoic anhydride, hexanoic anhydride, heptanedioic anhydride, and octandioic anhydride etc., and the adopted dithio-dihydrazides refer to those above mentioned.
The preparation of multiple modified derivatives of gelatin represented by above mentioned general formula (VI) includes two processes: chemical modification mode (B) i.e. carboxylation on the side chain amino group of gelatin through amide bond, and chemical modification mode (C) i.e. thiolation on the side chain carboxyl of gelatin. The commonly adopted preparation method is divided into two steps: (1) to dissolve gelatin in warm water to make aqueous solution (usually at 30° C.), then adjust the pH value of solution to subacidity (usually 4.75). After that, add in a certain amount of dithio-dihydrazide, stir and dissolve, then add in a certain amount of 1-ethyl-3-(3-dimethylamino propyl) carbodiimide hydrochloride, and at the same time add in appropriate amount of acid solution continuously (e.g. hydrochloric acid) to maintain the pH value of reaction solution at 4.75; Finally under the condition of alkalescence (usually the pH value is 8˜10), add in reduce agents such as hydroxyy thiol, dithiothreitol or sodium borohydride etc. to reduce the disulfide bond into free thiol, the impurities are purified by dialysis under acid condition. Freeze drying to get the intermediate. (2) to dissolve the intermediate into warm water to make aqueous solution under the protection of inert gas (e.g. Nitrogen etc.) (usually at 30° C.), then adjust the pH value of solution to alkalescence (usually 8˜10). After that, add in diacid anhydride, stir to react for a certain time, add in appropriate amount of aqueous alkali solution (e.g. sodium hydroxide) to maintain the reaction solution as alkalescence at the same time. Finally, purifying by dialyze the reaction solution under acid condition, and then freeze drying to get the multiple modified derivatives of gelatin represented by general formula (VI) of this invention. The adopted diacid anhydride includes butanedioic anhydride, glutaric anhydride, and hexanedioic anhydride etc., and the adopted dithio-dihydrazides refer to those above mentioned.
The preparation of multiple modified derivatives of gelatin represented by above mentioned general formula (VII) and (X) includes three processes: chemical modification mode (B) i.e. carboxylation on the side chain amino group of gelatin through amide bond, chemical modification mode (C) i.e. thiolation on the side chain carboxyl of gelatin, and chemical modification mode (D) i.e. thiolation after carboxylation on the amino group of gelatin through amide bond. The commonly adopted preparation method is divided into two steps: (1) to dissolve gelatin in warm water to make aqueous solution (usually at 30° C.), then adjust the pH value of solution to alkalescence (usually 8˜10). After that, add in diacid anhydride, stir to react for a certain time, add in appropriate amount of aqueous alkali solution (e.g. sodium hydroxide) to maintain the reaction solution as alkalescence at the same time. Finally, dialyze and purify the reaction solution, and then freeze drying to get the intermediate. (2) to dissolve the intermediate into warm water to make aqueous solution or directly use the unpurified intermediate solution (usually at 30° C.), then adjust the pH value of solution to subacidity (usually 4.75). After that, add into a certain amount of dithio-dihydrazide, stir and dissolve, then add a certain amount of 1-ethyl-3-(3-dimethylamino propyl) carbodiimide hydrochloride, and at the same time add into appropriate amount of acid solution continuously (e.g. hydrochloric acid) to maintain the pH value of reaction solution at 4.75; Finally under the condition of alkalescence (usually the pH value is 8˜10), add in reduce agents such as hydroxyy thiol, dithiothreitol or sodium borohydride etc. to reduce the disulfide bond into free thiol, the impurities are removed by dialysis and purification under acid condition. Freeze drying to get the product. If there is an overdose of 1-ethyl-3-(3-dimethylamino propyl) carbodiimide hydrochloride used in the reaction, then all carboxyls are modified into thiol, and the product is the multiple modified derivatives of gelatin represented by general formula (VII) of this invention; If there is less 1-ethyl-3-(3-dimethylamino propyl) carbodiimide hydrochloride used in the reaction, then only part of carboxyls are modified into thiol, and the product is the multiple modified derivatives of gelatin represented by general formula (X) of this invention. The adopted diacid anhydride includes butanedioic anhydride, glutaric anhydride, and hexanedioic anhydride etc., and the adopted dithio-dihydrazides refer to those above mentioned.
The preparation method for multiple modified derivatives of gelatin represented by above mentioned general formula (VIII) is substantially the same with that for multiple modified derivatives of gelatin represented by above mentioned general formula (VI), as long as adding anhydride and diacid anhydride simultaneously in the step (2) for preparation of multiple modified derivatives of gelatin represented by above mentioned general formula (VI).
The preparation method for multiple modified derivatives of gelatin represented by above mentioned general formulas (IX) and (XI) is substantially the same with that for multiple modified derivatives of gelatin represented by above mentioned general formulas (VII) and (X), as long as adding anhydride and diacid anhydride simultaneously into the step (2) for preparation of multiple modified derivatives of gelatin represented by above mentioned general formulas (VII) and (X). If there is an overdose of 1-ethyl-3-(3-dimethylamino propyl)carbodiimide hydrochloride used in the reaction, then all carboxyls are modified into thiols, and the product is the multiple modified derivatives of gelatin represented by general formula (IX) of this invention; if there is less 1-ethyl-3-(3-dimethylamino propyl)carbodiimide hydrochloride used in the reaction, then only part of carboxyls are modified into thiol, and the product is the multiple modified derivatives of gelatin represented by general formula (XI) of this invention.
The novel multiple modified derivatives of gelatin of this invention has at least one side chain free thiol, which can be re-oxidized to form disulfide bond under certain conditions. Moderate oxidants such as oxygen, low concentration of peroxide, iodine, trivalent iron ion etc., all can transform the free thiol into disulfide bond, thereby prepare crosslinked gelatin material. Its general preparation method is that the aqueous solution of multiple modified derivatives of gelatin of this invention is oxidized into disulfide bond crosslinked material using air under neutral or alkalescence conditions; or oxidized into disulfide bond crosslinked material using low concentration of stronger oxidants such as hydrogen peroxide or trivalent iron ion etc under weak acid or acid conditions.
The crosslinked material of multiple modified derivatives of gelatin of this invention can also be prepared through cross-linking between multiple modified derivatives of gelatin of this invention and thiol-reactive crosslinker. The thiol-reactive functional group adopted in this invention includes maleimide, vinyl sulfone, α,β-unsaturated acrylic ester, α,β-unsaturated methyl acrylic ester, halo-propionic ester, halo-propionamide, disulfo-pyridine, N-hydroxyl succimide activated ester etc. Wherein, maleimide, vinyl sulfone, iodo-propionic ester, iodo-propionamide, disulfo-pyridine etc functional groups have high thiol-reactivity. These reactions can be divided into three classes: (1) the addition reaction between thiol and active unsaturated double bond, wherein the functional groups belonging to this reaction contain maleimide, vinyl sulfone, α,β-unsaturated acrylic ester, α,β-unsaturated methyl acrylic ester etc. (2) The substitution reaction between thiol and active alkylogen, wherein the functional groups belonging to this reaction contain iodo-propionic ester, bromo-propionic ester, chloro-propionic ester, iodo-propionamide, iodo-propionamide, chloro-propionamide, and disulfo-pyridine etc. (3) the last class is thioesterification reaction, and the functional groups of this reaction contain activated esters of various carboxylic acids e.g. N-hydroxyl succimide activated ester etc. Take the multiple modified derivatives of gelatin represented by general formula (V) of this invention as an example, the reaction equations between sulfhydryl and these functional groups are as follows:

The thiol-reactive crosslinker adopted by this invention is polyethylene glycol (abbr. PEG) derivatives with at least two above mentioned functional groups e.g. two-arm, three-arm, four-arm, eight-arm or multiple-arm PEG derivatives, and they have the following typical chemical structures:

Wherein, F1, F2, F3, F4, F5, F6, F7 and F8 are above mentioned thiol-reactive functional groups e.g. maleimide, vinyl sulfone, α,β-unsaturated acrylic ester, α,β-unsaturated methyl acrylic ester, halo-propionic ester, halo-propanamide, disulfo-pyridine, N-hydroxyl succimide etc., and they may have totally or partially same, or totally different chemical structures. PEG refers to the segmer having repetitive units of CH2CH2O whose molecular weight is from 100 to 1000000.
Take two-arm PEG as an example, the common crosslinker adopted by this invention contains PEG di-maleimide, PEG divinyl sulfone, PEG di-acrylic ester, PEG di-acrylamide, PEG di-halo-propionic ester, PEG di-halo-propanamide, PEG di-dithio-pyridine, and PEG di-N-hydroxyl succimide etc. There chemical structures are as follows:

The general preparation method, adopted by this invention, for novel crosslinked material made of the multiple modified derivatives of gelatin crosslinked by thiol-reactive crosslinker is to make the multiple modified derivatives of gelatin of this invention into aqueous solution or mixed aqueous solution, adjust the pH value of solution to be neutral, then add in above mentioned thiol-reactive crosslinker aqueous solution, after mixed uniformly, keep standing at room temperature for a moment and then get the gel i.e. crosslinked material. Take above mentioned two-arm PEG derivative crosslinker and the multiple modified derivatives of gelatin represented by general formula (V) of this invention as an example, the prepared crosslinking material has the following chemical structure:

Similar to the multiple modified derivatives of gelatin represented by general formula (V) of this invention, the multiple modified derivatives of gelatin represented by general formula (VI), (VII), (VIII), (IX), (X) and (XI) of this invention also contain at least one thiol, and the same above mentioned crosslinking mode can also be used to prepare crosslinked material. In addition, co-crosslinking with a multi-arm PEG derivative crosslinker can also be adopted to prepare the crosslinked material made of multiple modified derivatives of gelatin of this invention. In addition, two or more above PEG derivative crosslinkers (e.g. two-arm PEG derivatives, three-arm PEG derivative cross-linker, four-arm PEG derivative crosslinker, eight-arm PEG derivative cross-linker etc.) can be adopted to prepare cross-linked material made of multiple modified derivatives of gelatin of this invention. The above mentioned preparation method, in combination with two or more above multiple modified derivatives of gelatin of this invention simultaneously, can be used to prepare the crosslinked material of gelatin.